Fluorescence immunoassays based on energy transfer have been taught by Lakowicz et al. in U.S. Pat. No. 5,631,169. A number of molecular species have been used for causing energy transfer from a donor molecule to an acceptor molecule. In particular, sandwich type immuno-complex formation can be used with this technique. For instance, this technique can be used for immunoassays based on changes in fluorescence lifetime with changing analyte concentration. However, this technique works with short lifetime dyes like fluorescein isothiocyanate (FITC) (the donor) whose fluorescence is quenched by energy transfer to Eosin (the acceptor). A problem or disadvantage of this technique is that short lifetimes are extremely difficult to measure. Moreover, the donors used in this invention have relatively short Stoke shifts, which could cause excitation signal to overlap the acceptor absorption thereby causing spurious signals, particularly when broad band emitters like LEDs are used as excitation sources.
Organo-metallic complexes can be used as donors without identifying suitable acceptors. However, there are a number of small molecules such as oxygen that also quench fluorescence of these complexes. For these reasons a number of problems exist when using some of the standard organo-metallic complexes for quenching experiments, measurements or immunoassay studies. Organo-metallic complexes, however, have the advantage of providing more suitable long lifetime based sensing and quenching. Consequently, assays based on this principle would require that the sample be purged of oxygen prior to analysis. This not only adds an additional sample preparation step, but also precludes one from analyzing samples that could be altered by such processing.
Accordingly, there is a substantial need for techniques and compositions of matter that allow for the use of a long lifetime donors to be used in conjunction with immunoassays based on the use of energy transfer. In addition, there is a need for donors that will allow for larger Stoke shifts, but not suffer from the limitation of quenching by molecules such as molecular oxygen. Furthermore, there is a need for techniques and compositions of matter that allow for the use of organo-metallic complexes that can be applied in the presence of changing concentration of collisional quenchers.
The above reference(s) and all other references cited in this application are incorporated in this application by reference. However, cited references or art are not admitted to be prior art to this application.